Enzyme assisted degradation of surface membranes of harvested fruits and vegetables

ABSTRACT

The present invention discloses that the water permeability across the surface membrane of harvested fruits and vegetables can be substantially increased by treating such surfaces with a degradation enzyme. The resulting products are not only more easily dehydrated but can be used to incorporate desirable substances into the interior of the treated fruit or vegetable, such as sweeteners, stabilizers, preservatives, flavor enhancers.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is directed to enzyme assisted degradation ofsurface membranes of unmacerated harvested fruits and vegetables. Inparticular, the present invention utilizes degradation enzymes such ascutinases, lipases, cellulases, pectinases, etc., to degrade one or moreof the water insoluble components which comprise part of the surfacemembrane of the fruits and vegetables. Preferably, the surface of thefruit or vegetable is sufficiently degraded so as to result in anincrease in water permeability across the surface membrane of at least50 percent as compared to untreated surfaces. The increased waterpermeability across the surface membrane permits more facile delivery ofsubstances such as flavorings, sweeteners, stabilizers, andpreservatives to the interior of the fruit or vegetable. Additionally,the increased water permeability allows for the more efficientdehydration of fruits and vegetables.

2. Related Art

The surface membranes of fruits and vegetables contain one or more typesof water insoluble components which significantly limit the permeabilityof water across the membrane. Accordingly, this membrane serves theuseful role of preventing the evaporation of liquids contained in fruitsand vegetables both prior to and after the harvesting of such fruits andvegetables. On the other hand, the production of dried fruits andvegetables require the evaporation (dehydration) of water from theinterior of harvested fruits or vegetables. Harvested fruits andvegetables are dried for a variety of reasons. First and most important,dehydration is a method of fruit preservation. Additionally, certaindried fruits and vegetables, i.e., raisins, prunes, apricots, sundriedtomatoes, etc., have desirable properties not present prior to drying.One means commercially utilized for drying fruits and vegetables is byosmosis. However, as noted above, the water insoluble componentscontained in the surface membranes of fruits and vegetables render thesurface not completely permeable to water. Thus, while the membranes ofcells of fruits and vegetables (below the surface membrane) areselectively permeable to molecules such as water and are thereforesusceptible to osmosis, the surface membranes of harvested fruits andvegetables limit osmosis as a means for effecting dehydration. Inparticular, such osmotic processes inherently require prolongeddehydration.

Additionally, the lack of significant water permeability across thismembrane also interferes with the osmotic transport of either natural orsynthetic substances into the interior of the fruit or vegetable. On theother hand, the delivery of substances, such as sweeteners, flavorenhancers, preservatives, stabilizers, etc., into the interior of suchproducts could provide beneficial results.

Certain chemicals such as organic solvents, including methanol,chloromethanes (chloroform, methylene chloride), etc., alkali metalhydroxides, etc., can be used to enhance the water permeability of thesurface membrane. However, the use of such chemicals is not desirablebecause traces of these chemicals are retained in the final product andthe subsequent ingestion of these chemicals could pose potential harmfulside effects. Worker exposure to these chemicals is undesirable andwaste treatment of these chemicals is hazardous and difficult.Furthermore, a growing concern among consumers regarding chemicals infood products makes these products less desirable.

While numerous degradation enzymes are known in the art with some beingcommercially available, these degradation enzymes are primarily knownfor their role in fruit and/or vegetable decay. On the other hand, somereferences disclose the use of enzymes on fruit and/or vegetables forthe purpose of providing enhanced protection for the treated product.

For example, European Patent Application No. 302 685A discloses the useof an auxin destroying material on the surface of fruit which reducesthe occurrence of russeting on the fruit. Auxin is defined by thisreference as chemicals, i.e., indole-3-acetic acid, etc., having commonbiological properties in plants such as stimulation of cell division,stimulation of shoot growth, control of vascular system differation,control of tissue culture differation, control of apical dominance, etc.The auxin destroying materials include microorganisms or compositionscontaining purified components thereof which can destroy the auxin bychemical degradation. Presumably, the auxin destroying materialgenerated by these microorganisms is an enzyme.

Likewise, U.S. Pat. No. 4,762,547 as well as U.S. Ser. No. 297,224 filedJan. 13, 1989 discloses the use of esterases, i.e., degradation enzymes,in combination with a biocide. These references disclose that when theesterase is used in conjunction with a biocide, which can be appliedeither separately or in combination, the esterase results in enhancedbiocide activity. U.S. Pat. No. 4,762,547 speculates that the esterasedecomposes the foliar wax (surface membrane) of the plant or theepidermal wax of insects, thereby permitting a larger amount of thebiocide to enter the treated plant or insect thus resulting in greaterbiocide activity. Implicit in these references is the fact that theesterase and biocide are applied to plants prior to harvesting.

However, none of these references disclose the use of esterases on thesurface membranes of harvested fruits and vegetables as a means forenhancing the water permeability across this surface.

Accordingly, it is an object of this invention to enhance the waterpermeability of the surface membrane of harvested fruits and vegetableswithout the use of chemicals which could pose potential harmful sideeffects if ingested. Such permeability should preferably be increased byat least 50 percent as compared to untreated surfaces.

It is a further object of this invention to facilitate the dehydrationof fruits and vegetables without the need for chemicals which act toenhance the surface membrane's water permeability.

It is still a further object of this invention to facilitate thedelivery of desirable substances to the interior of the fruit orvegetable.

These and other objects are achieved by the present invention asevidenced by the attached summary of the invention, detailed descriptionof the invention, examples, and claims.

SUMMARY OF THE INVENTION

The present invention is directed to methods for increasing the waterpermeability of surface membranes of unmacerated harvested fruits andvegetables. The present invention is also directed to the resultingproducts obtained from such methods. Thus, in its first method aspect,the present invention is directed to a method for increasing the waterpermeability across the surface membrane of harvested fruits andvegetables wherein said surface membrane contains one or more types ofwater insoluble components. The method comprises exposing this surfacemembrane to a sufficient concentration of a degradation enzyme capableof degrading at least one of said water insoluble components for asufficient period of time so as to provide an increase in waterpermeability across the membrane of at least 50 percent as compared tountreated fruit or vegetable.

In another method aspect, the present invention is directed to a methodfor preparing dehydrated fruits or vegetables from harvested fruits andvegetables wherein said harvested fruits and vegetables have a wateractivity (a_(w)) of greater than 0.80 and further have a surfacemembrane which contains one or more types of water insoluble componentswhich method comprises exposing the surface membrane of the harvestedfruit or vegetable to a sufficient concentration of a degradation enzymecapable of degrading at least one of the water insoluble components fora sufficient period of time so as to provide an increase in waterpermeability across the membrane of at least 50 percent as compared tothe untreated fruit or vegetable; and dehydrating the fruit or vegetableproduct so produced under conditions sufficient to reduce its a_(w) to0.80 or less.

In another method aspect, the present invention is directed to a methodof transporting a natural or synthetic substance into the interior of aharvested fruit or vegetable having a surface membrane which containsone or more types of water insoluble components which comprises exposingthe surface membrane of the fruit or vegetable to a sufficientconcentration of a degradation enzyme capable of degrading at least oneof water insoluble components for a sufficient period of time so as toprovide an increase in water permeability across the membrane of atleast 50 percent as compared to the untreated fruit or vegetable; andexposing the product so produced with an aqueous solution containingsufficient quantities of natural or synthetic substance(s) for asufficient period of time so as to allow the substance(s) to beosmotically transported to the interior of the product.

In its first product aspect, the present invention is directed to amodified harvested fruit or vegetable product having a surface membranecontaining water insoluble components wherein the membrane has a waterpermeability across this membrane of at least 50 percent greater thanthe unmodified product and wherein the modified product is free ofpermeability enhancing chemicals.

In its second product aspect, the present invention is directed to themodified harvested fruit or vegetable product described above and whichadditionally contains in the interior thereof a quantity of one or morenatural substances in excess of that occurring naturally in theunmodified product.

In yet another product aspect, the present invention is directed to themodified harvested fruit or vegetable product described above and whichadditionally contains in the interior thereof one or more syntheticsubstances. Preferably, the synthetic substance is selected from thegroup consisting of sweeteners, flavor enhancers, preservatives,stabilizers, etc.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to enzyme assisted degradation ofsurface membranes of harvested fruits and vegetables. In particular,surface membranes of fruits and vegetables contain, in part, one or moretypes of water insoluble components which significantly limit thepermeability of water across this membrane. As a result, the dehydrationof or the osmotic delivery of substances into the interior of fruits andvegetables is impaired. Methods disclosed by this invention result inmodified fruit and vegetable products having substantially greaterpermeability to water than unmodified products. As a result, themodified fruit and vegetable products of this invention not only permitthe more facile dehydration of these products, such as in thepreparation of dried fruits and vegetables, but also permit the osmoticdelivery of substances to the interior of these products. Moreover, theproducts of this invention are free of permeability enhancing chemicals.

Prior to discussing this invention in detail, the following terms willbe defined:

"Water insoluble components"--refer to those components which comprisepart of the surface membrane of fruits and/or vegetables therebyrendering this surface membrane not completely permeable to water. Suchcomponents do not have any significant solubility in water and cancomprise polymers such as cutin, cellulose, pectin, etc., as well aswater insoluble materials such as triglyercides, wax esters, etc. Otheradditional water insoluble components are well known in the art.

"Degradation enzymes"--refer to enzymes capable of degrading at leastone type of water insoluble components. Suitable degradation enzymesinclude lipase, cutinase, cellulase, pectinase, etc. Lipase sourcesinclude those originating in the genera Candida, Pseudomonas, Rhizopus,Aspergillus, Mucor, etc. Such lipases are capable of degrading the waterinsoluble triglycerides which can comprise part of the surface membrane.Commercially available products can be used for the present invention.

Cutinase sources include those originating in the genera Pseudomonas,Fusarium, Botrytis, Ulocladium, etc. See, for instance, "Cutinases fromFungi and Pollen", P. E. Kolattukudy, pages 472-504 and which isincorporated herein by reference. A particularly preferred cutinase isone prepared from the genus Pseudomonas mendocina ATCC 53552, theisolation and specific amino acid sequence of which is described in U.S.Ser. No. 107,092, filed Oct. 19, 1987, entitled "Novel Hydrolase andMethod"; as well as U.S. Ser. No. 297,224, filed Jan. 13, 1989 andentitled "Enzymes as Agricultural Chemical Adjuvants", the disclosure ofboth of which are incorporated herein by reference in their entirety.Other cutinases can include commercially available products. Suchcutinases are capable of degrading the water insoluble cutin polymerwhich can comprise part of the surface membrane of the fruit orvegetable.

Cellulase sources include those originating in the genera Trichoderma,Penicillum, Aspergillus, Clostridium, etc. Additional cellulases caninclude commercially available products. Such cellulases are capable ofdegrading the water insoluble cellulose polymer comprising part of thesurface membrane of the fruit or vegetable.

Lipase sources include those originating in the genera Staphylococcus,Candida, Rhizopus, etc. Additional lipases can include commerciallyavailable products. Such lipases are capable of degrading the waterinsoluble gylcerol components comprising part of the surface membrane ofthe fruit or vegetable.

Pectinase sources include those originating in the genera Rhizopus,Penicillium, Aspergillus. etc. Additional pectinases can includecommercially available products. Such pectinases are capable ofdegrading the water insoluble pectin components comprising part of thesurface membrane of the fruit or vegetable.

Also included within the term "degradation enzyme" are two or moredegradation enzymes which have been combined, i.e., a lipase combinedwith a cutinase, etc. Such combinations may provide for degradationcapabilities against two or more types of water insoluble componentswhich may be contained in the surface membrane.

"Non-ionic surfactant"--refers to surfactants which have neither acationic nor an anionic group. Such non-ionic surfactants includeTriton™, especially Triton™ X-100, Span™, Tween™, sucrose esters, alkylglucosides, etc. The particular non-ionic surfactant employed is notcritical provided it does not interfere with the enzyme activity of thedegradation enzyme.

"Natural substances"--refer to substances which are indigenous to thatfruit or vegetable and includes substances such as sugars, antibiotics,etc. produced by that particular fruit or vegetable.

"Synthetic substances"--refer to substances which are not indigenous tothat fruit or vegetable but which if included in the interior of thefruit and/or vegetable would provide beneficial results. Such syntheticsubstances include sweeteners, flavor-enhancers, preservatives,stabilizers, etc. Preferably, the synthetic substance is one which isnaturally produced albeit not by that fruit or vegetable, i.e., asubstance produced by either an organism or by a different fruit, plantor vegetable.

The term "a_(w) "--refers to water activity and is a measure of thewater content in the fruit or vegetable. Fresh fruits typically havewater contents greater than 90%, with water activities (a_(w)) of about0.97. Fruit dried using conventional technology lowers its water contentto about 20%, with an a_(w) from about 0.72 to about 0 80. It isparticularly desirable that the a_(w) of the dried product be lowered to0.80 or less. This is because bacteria, yeasts, and mold do not grow atan a_(w) below 0.91, 0.88, and 0.80 respectively. Thus lowering thea_(w) of the dried product to 0.80 or less, provides for a product notsusceptible to spoilage by bacteria, yeasts, and molds. In general,freshly harvested fruits and vegetables to be used in the methods ofthis invention should have an a_(w) of greater than 0.80; preferablygreater than 0.90; and even more preferably greater than 0.95.

"Permeability enhancing chemicals"--refer to certain chemicals, such asorganic solvents, e.g., methanol, chloroform, methylene chloride, etc.,alkali metal hydroxides, including sodium hydroxide and potassiumhydroxide, which when exposed to the surface membrane enhance its waterpermeability. Characteristic of such permeability enhancing chemicals isthe fact that these chemicals are not naturally produced and may posepotential harmful side effects if ingested. On the other hand, as usedherein, the term "permeability enhancing chemicals" does not includedegradation enzymes which are naturally produced enzymes, i.e.,proteins, which can be transformed when ingested into useful products,i.e., polypeptides and amino acids including essential amino acids.

The methods of the present invention utilize enzyme assisted degradationof one or more of the water insoluble components found in the surfacemembranes of fruits and vegetables as a means of enhancing the waterpermeability across this membrane. In general, the surface membrane of aharvested fruit or vegetable is exposed to a sufficient concentration ofa degradation enzyme for a sufficient period of time so as to achievethe desired increase in water permeability across the membrane. Becausethe degradation enzyme's activity is stable over an extended period oftime, e.g., for several days at pH 7, it would be anticipated that theconcentration of degradation enzyme required to achieve the desiredincrease in permeability would be inversely proportional to the exposuretime. That is to say, if a short exposure time is employed, then higherconcentrations of degradation enzyme would be required to achieve thedesired permeability. Conversely, if longer exposure times are employed,then the concentration of the degradation enzyme could be proportionallyreduced to still arrive at the desired result. While the abovecorrelation is generally true for most enzymes, certain enzymes such ascutinase derived from Pseudomonas putida can exhibit reduced activity atconcentrations above 0.3 mg/ml. While not being limited to any theory,it is believed that this result arises because of concentrationdependent aggregation effects arising in this enzyme. However, any suchaggregation can be diminished by using art recognized techniques such asthe use of detergents, etc.

With regard to exposure times, the increase in water permeability willinitially be linear with exposure time and then asymptotically approachthe maximum increase possible. Thus, while the above correlation isgenerally true for exposures times falling on the linear portion ofactivity, it is not true for the non-linear (asymptotic) portion. In anyevent, it is well within the skilled artisan's ability to select theappropriate concentration of degradation enzyme and the appropriateexposure time to arrive at the desired increase in water permeability.

In a preferred embodiment, the degradation enzyme is generally employedat a concentration of at least about 0.01 mg/ml; more preferably, theconcentration of degradation enzyme is from about 0.05 mg/ml to about1.0 mg/ml; and even more preferably at a concentration of from about 0.1mg/ml to about 0.5 mg/ml.

Also, in a preferred embodiment, the exposure time of the degradationenzyme to the surface membrane of the fruit or vegetable is generally atleast one hour; more preferably, the exposure time is from about fourhours to about twenty-five hours; and even more preferably from aboutten hours to about twenty hours.

The degradation enzyme is generally exposed to the surface membrane ofthe fruit or vegetable at a temperature sufficiently high for the enzymeto exhibit an acceptable level of activity but not so high as to causethe enzyme to denature. In general, temperatures of from about 10° C. toabout 50° C. are acceptable while temperatures of from about 20° C. toabout 40° C. are preferred.

In general, the fruit or vegetable is exposed to a sufficientconcentration of the degradation enzyme for a sufficient period of timeso as to result in an increase in water permeability of at least 50%over untreated fruit or vegetable. Preferably, if the treated fruit orvegetable is to be used to prepare a dried product, then the increase inpermeability should be at least about 200%, and more preferably, atleast about 1000%. Most preferably, the increase in permeability shouldbe a large as possible in order to speed the dehydration process. On theother hand, if the treated fruit or vegetable is to be used in theprocess which incorporates natural or synthetic substances into theinterior thereof, than preferably the increase in permeability should befrom at about 50% to about 500% in order to retain as much of theproduct's original appearance and texture as is possible.

Preferably, the degradation enzyme is employed in an aqueous solutionwhich can be so selected so as to provide optimum conditions for enzymeactivity. Thus factors such as pH, stabilizers, temperatures, buffercontent, etc., can be readily maintained or incorporated into theaqueous solution. For example, the pH employed in combination with thedegradation enzyme can range from about 4 to 11, with a pH of 10 beingpreferred when the cutinase is isolated from Pseudomonas putida. Anappropriate pH can readily be maintained in an aqueous solution by theappropriate choice of buffers. Additionally, the aqueous solution canalso contain any necessary substrate required for enzyme activity (ifnot contained in the vegetable or fruit) as well as stabilizers,scavengers, etc.

In a preferred embodiment, the aqueous solution additionally contains anon-ionic surfactant. While this non-ionic surfactant has little or noeffect on dehydration by itself, the combination of the degradationenzyme and the non-ionic surfactant synergistically enhances the amountof water insoluble component degraded by the degradation enzyme (asmeasured by an increase in dehydration which correlates to surfacemembrane's water permeability). Without being limited to any theory,this synergy appears may be due to the non-ionic surfactant aiding thedegradation enzyme in penetrating the surface membrane, which makes thewater insoluble components more available to the degradation enzyme.When so used, the non-ionic surfactant is generally employed in anamount sufficient to enhance the degradation activity of the degradationenzyme. Preferably, the amount of nonionic surfactant employed rangesfrom about 0.005% to about 0.5% and more preferably, from about 0.01% toabout 0.10%.

As before, the surface membrane of the fruit or vegetable is exposed toa sufficient concentration of degradation enzyme for a sufficient periodof time so as to result in the desired increase in water permeability.At this time, this product is then subjected to dehydration if a driedproduct is desired. One method of dehydrating is by osmosis. In osmosis,an aqueous solution containing a sufficient concentration of solute isexposed to the surface membrane of the fruit or vegetable. The amount ofsolute contained in solution must be greater than its concentration inthe fruit or vegetable. Under such conditions, osmosis occurs, i.e., thesolute is transported into the fruit or vegetable and water istransported from the fruit or vegetable into the aqueous solution with anet loss of water in the fruit or vegetable. Osmosis will continue untilequal concentrations of solute are found on both sides of the surfacemembrane. Thus, by merely selecting the concentration of solute, one canselect the amount of water removed from the fruit or vegetable.Preferably, the solute employed is a natural product such as a sugar,i.e., sucrose, glucose, fructose, etc., glycerol, etc., which, as above,is incorporated into the product. In addition, the solution can containmaterials such as vitamins, minerals, etc., which during the course ofosmotic dehydration would also be incorporated into the product.

The osmotic conditions selected are not critical as long as they aresufficient to permit osmosis to proceed. For example, the time for whichosmosis is allowed to proceed is not critical and is generally governedby the desired degree of dehydration, i.e., osmosis is allowed toproceed until the desired degree of dehydration is reached and thenterminated. Likewise, the temperature that the osmosis is conducted atis not critical but temperatures of from about 10° C. to about 40° C.are preferred. Solute concentrations also are not critical but should beselected so as to allow osmosis to continue to at least the point wherethe desired degree of dehydration has occurred. Additionally, soluteconcentration is governed by the solute's solubility in solution. Soluteconcentrations up to its maximum solubility in solution can be used.Lastly, the solute employed must be permeable to the surface membrane.Solutes such as sugars, glycerol, aspartame, vitamins, etc., aresuitable. Other suitable solutes are well known in the art. The pH ofthe solution is also not critical and can range from about 4 to about11, although certain pH' s may be preferred. For example, when cutinaseisolated from Pseudomonas putida is used as the degradation enzyme, a pHof about 7-10 is preferred with of 10 being optimum for dehydration(osmosis)--possibly due to residual cutinase activity which has anoptimal pH of about 10.

Dehydration is generally allowed to continue until the a_(w) of theproduct is 0.80 or less. After the desired degree of dehydration hasbeen obtained, the resulting dehydrated product is removed from theosmotic conditions and then is preferably dried to remove residualmoisture. The particular drying method is not critical and any suitabledrying means can be used, i.e., evaporation, blotting, etc.

Another suitable dehydration method is evaporation which includessun-drying, heating, etc. For example, in sun-drying, the fruit orvegetable is exposed to sunlight for a suitable length of time to allowa sufficient amount of moisture to be removed by evaporation from theproduct. Suitable conditions are well known in the art. In any event, byincreasing the surface membrane's permeability, it is possible toincrease the speed of dehydration whether dehydration is by osmosis,evaporation, etc.

When the product obtained by exposing the surface of the fruit orvegetable to an degradation enzyme is intended to be used for theincorporation of a natural or synthetic substance into the interior ofthe product, the osmotic conditions described above are equallypertinent here with the following exceptions. Firstly, while theduration of osmosis is still not critical, it is neverthelesssubstantially shorter than the duration of osmosis in hydration becausesignificant dehydration is not intended and, in fact, not desired. Thatis because significant dehydration, i.e., water loss of 10% or more fromthe product, can result in loss of the original appearance and textureof the treated fruit or vegetable. In order to minimize dehydration andchange in product appearance and texture, the increase in waterpermeability across the surface membrane should be kept in the range ofat least 50% to about 500% and preferably from about 100% to about 400%as compared to the untreated fruit or vegetable. In any event, the timeemployed for osmosis is generally a duration sufficient to allow theosmotic transport of the desired quantity of natural of syntheticsubstance into the interior of the product.

Secondly, the concentration of solute is generally less than in osmoticdehydration in order to minimize dehydration. For example, soluteconcentrations slightly higher than the concentrations of the solute inthe fruit or vegetable, at most about 10 mg/ml higher, allow forincorporation of the solute into the interior of the product withoutsignificant dehydration. It is also noted that the concentration of thesolute can differ for natural and synthetic substances. In particular,for a natural substance, it is necessary to employ a higherconcentration of this substance in the aqueous solution than is found inthe product in order for this substance to be transported into theproduct by osmosis. On the other hand because a synthetic substance isby definition a substance not contained in the product, anyconcentration of this substance in solution will be greater than that inthe product and osmosis will proceed. Selecting the appropriateconditions for osmotically transporting a natural or synthetic substanceinto the interior of a fruit or vegetable product treated with adegradation enzyme is well within the capabilities of the skilledartisan. The product prepared by osmotically transporting the natural orsynthetic substance into the interior of the fruit or vegetable is bynecessity free of permeability enhancing chemicals because suchchemicals were not used.

The following examples are offered to illustrate the invention andshould not be construed in any way as limiting the scope of thisinvention.

EXAMPLES

In the following examples, recitation of percentages in solution referto weight percentages. Recitation of tomatoes refer to cherry tomatoeswhich were purchased locally and selected for their similarity in color,weight and skin quality. Typically groups of five (having a total massof 100-150 grams) were rinsed and washed and then used in the examples.In Examples 1-7, cutinase refers to the cutinase enzyme isolated fromPseudomonas putida described earlier.

EXAMPLE 1

Tomatoes were incubated in an aqueous solution containing 100 mM glycinebuffer and 0.05% sodium azide which optionally contained 0.18 mg/mlcutinase at various pH's for 18 hours at 37° C. They were then washedwith water, blotted dry on a paper towel, and placed in an aqueousdehydrating solution containing 50% sucrose, 0.05% sodium azide at aratio of 1.8 ml of solution per gram of tomato. After 408 hours at roomtemperature at about pH 7, they were removed, blotted dry, and weighed.[In this and in subsequent examples, solutions containing cutinase areindicated by + (plus); solutions not containing cutinase are indicatedby - (minus)]

    ______________________________________                                        pH           cutinase % weight loss                                           ______________________________________                                        4.6          -        13                                                      4.6          +        17                                                      7.5          -        18                                                      7.5          +        21                                                      10.0         -        21                                                      10.0         +        33                                                      ______________________________________                                    

The above data demonstrates that incubation with cutinase increases the% weight loss at all pH's tested. However, the above data alsodemonstrates that higher pH increases the % of weight loss, and the twoexhibit a concerted effect.

EXAMPLE 2

A. Tomatoes were incubated in an aqueous solution containing 0.16 mg/mlcutinase, 100 mM glycine, 0.05% sodium azide at pH 10, for 18 hours at22° C. They were then washed with water blotted dry on a paper towel,and placed in an aqueous dehydrating solution containing 50% glycerol,100mM glycine, 0.05% sodium azide buffered to various pH's at a ratio of1.8 ml/g tomato. After 508 hours at 22° C., they were removed, blotteddry, and weighed.

    ______________________________________                                        pH           cutinase % weight loss                                           ______________________________________                                        7.0          +        33                                                      10.0         +        35                                                      ______________________________________                                    

B. In a similar treatment as A above, tomatoes were incubated in thesame aqueous solution optionally containing cutinase (0.16 mg/ml) butfor 18 hours at 37° C. They were then washed with water, blotted dry andplaced in the same dehydrating medium under the same conditions but atvarying pH's. After 144 hours at 22° C., the tomatoes were removed,blotted dry, and weighed.

    ______________________________________                                        pH           cutinase % weight loss                                           ______________________________________                                        8.0          -        12                                                      8.0          +        16                                                      10.0         -        11                                                      10.0         +        21                                                      ______________________________________                                    

C. In a similar treatment as A above, tomatoes were incubated for thesame length of time in the same aqueous solution containing cutinase atthe same temperature. They were then washed with water, blotted dry on apaper towel and placed in an aqueous dehydrating solution containing 25%glucose, 100 mM glycine, 0.05% sodium azide, at a ratio of 1.8 mlsolution per gram of tomato. After 508 hours at 22° C., the tomatoeswere removed, blotted dry, and weighed.

    ______________________________________                                        pH           cutinase % weight loss                                           ______________________________________                                        7.0          +        19                                                      8.0          +        17                                                      9.0          +        12                                                      10.0         +        21                                                      ______________________________________                                    

EXAMPLE 3

Tomatoes were incubated for 18 hours at 37° C. in 100 mM glycine, pH 10,0.5% sodium azide and two different cutinase concentrations. They werethen washed, blotted dry, and placed in an aqueous dehydrating solutioncontaining 50% sucrose, 100 mM glycine, 0.05% sodium azide at pH 10 andat 22° C. A ratio of 1.8 ml/gram of tomato was maintained throughout.After 408 hours, they were removed and weighed.

    ______________________________________                                        cutinase concentration                                                                         % weight loss                                                ______________________________________                                        0.18 mg/ml       33                                                           0.36 mg/ml       15                                                           ______________________________________                                    

Without being limited by an theory, it is believed that the decrease inweight loss with increasing cutinase concentration could be the resultof concentration dependent aggregation effects.

EXAMPLE 4

Tomatoes were incubated with 0.16 mg/ml cutinase, 100 mM glycine, pH 10,0.05% sodium azide for 18 and 40 hours at 37° C. They were then washed,blotted dry and placed in an aqueous dehydrating solution containing 100mM glycine, 50% glycerol, 0.05% azide solution at pH 10 and a ratio of1.8 ml of dehydrating solution per gram of tomato was maintainedthroughout. After 144 hours at 22° C., they were removed and weighed.

    ______________________________________                                        18 hours     21% weight loss                                                  40 hours     20% weight loss                                                  ______________________________________                                    

The above data indicates that under these conditions, the maximumincrease in permeability to the surface membrane was reached within thefirst 18 hours of exposure to cutinase.

EXAMPLE 5

Tomatoes were incubated in an aqueous solution containing 100 mMglycine, 0.05% sodium azide and optionally 0.16 mg/ml cutinase and 0.01or 0.02% sodium dodecylsulfate at pH 10 and at 37° C. for 18 hours.After incubation, the tomatoes were washed, blotted dry with a papertowel and added to an aqueous dehydrating solution containing 50%glycerol, 100 mM glycine, 0.05% sodium azide at pH 10 and at 22° C. for144 hours at a ratio of 1.8 ml of solution per gram of tomato.Afterwards, the tomatoes were removed, blotted dry and weighed. Thedifferent solutions used are set forth in Table II below:

                  TABLE II                                                        ______________________________________                                                  Solutions                                                                     I   II       III   IV     V   VI                                    ______________________________________                                        cutinase.sup.a                                                                            -     +        -   -      +   +                                   0.01% SDS.sup.b                                                                           -     -        +   -      +   -                                   0.02% SDS.sup.b                                                                           -     -        -   +      -   +                                   ______________________________________                                    

The results of this test are set forth in Table III below.

                  TABLE III                                                       ______________________________________                                                Solutions                                                                     (% weight loss)                                                       hours     I     II        III IV      V   VI                                  ______________________________________                                        144       11    21        12  13      21  22                                  ______________________________________                                    

The above results show that an ionic surfactant does not have anysignificant effect on the dehydration of the tomatoes.

EXAMPLE 6

The procedure set forth in Example 5 was followed except that anon-ionic surfactant, TRITON X-100, was used in place of SDS and thetomatoes were weighed during a time course of 16, 40 and 144 hours. Thedifferent solutions used are set forth in Table IV below:

                  TABLE IV                                                        ______________________________________                                                  Solution                                                                      VII   VIII    IX     X    XI   XII                                  ______________________________________                                        cutinase.sup.a                                                                            -       +       -    -    +    +                                  TRITON X-100                                                                              -       -       +    -    +    -                                  (0.01%)                                                                       TRITON X-100                                                                              -       -       -    +    -    +                                  (0.02%)                                                                       ______________________________________                                    

The results of this test are set forth in Table V below:

                  TABLE V                                                         ______________________________________                                               Solution                                                                      (% weight loss)                                                        hours    VII    VIII      IX  X      XI  XII                                  ______________________________________                                         16      --     --         2   1     24  30                                    40      --     --         5   3     37  41                                   144      11     21        14  11     40  45                                   ______________________________________                                    

The above data demonstrates that within 40 hours, over 40% of the weightof the tomatoes treated with solution XII was lost, while the tomatoestreated with solution X lost 3% of their weight in the same amount oftime. Likewise, tomatoes treated with solution VIII lost 21% of theirweight but over 144 hours. While the non-ionic surfactant had littleeffect on dehydration by itself, the above data also demonstrates thatthere is more than an additive effect when both the cutinase andnon-ionic surfactant are used together.

By following the procedures set forth above in Examples 1-7 above, otherfruits and vegetables such as grapes, apricots, plums, apples, etc.,could be substituted for tomatoes to achieve a dehydrated product.Likewise, other degradation enzymes, such as lipases, pectinases,cellulases, etc., could be substituted for cutinase to achieve adehydrated product.

EXAMPLE 8

Tomatoes are rinse, weighed, and incubated in an aqueous solutioncontaining 100 mM glycine, 0.05% sodium azide and 0.10 mg/ml ofpectinase isolated from Aspergillus at pH 10 for 3 hours at 22° C. Thetomatoes are then washed with water, blotted dry, and placed in anaqueous solution containing 1% of aspartame (a sweetner), 100 mMglycine, and 0.05% sodium azide at a ratio of 1.8 ml of solution to gramof tomato. After 48 hours, the tomatoes are removed and blotted dry. Theabove procedure permits the incorporation of a sweetner into the tomatowith minimal dehydration. By following the above procedure, othersubstances such as preservatives, stabilizers, color enhancers, etc.,could be substituted for tomatoes to achieve a product having thissubstance incorporated into the interior thereof. Likewise, otherdegradation enzymes, such as lipase, cellulase, cutinase, etc., could besubstituted for pectinase to achieve a modified product.

What is claimed is:
 1. A method for increasing the permeability of wateracross the surface membrane of unmacerated harvested fruits andvegetables wherein said surface membrane contains one or more types ofwater insoluble components selected from the group consisting of cutin,cellulose, pectin, triglycerides and waxy esters, said method comprisesexposing said surface membrane to a composition comprising a sufficientconcentration of a degradation enzyme selected from the group consistingof lipase, cutinase, cellulase, and pectinase and a sufficientconcentration of a non-ionic surfactant so as to enhance the activity ofsaid degradation enzyme for a sufficient period of time so as to providefor an increase in water permeability across said membrane of at leastfifty percent as compared to the water permeability of said harvestedfruits and vegetables prior to exposure to said composition.
 2. Themethod according to claim 1 wherein the increase in water permeabilityacross said membrane is at least two hundred percent.
 3. The methodaccording to claim 2 wherein the increase in water permeability acrosssaid membrane is at least one thousand percent.
 4. The method accordingto claim 1 wherein said degradation enzyme is contained in an aqueoussolution.
 5. The method according to claim 4 wherein the concentrationof said degradation enzyme in said aqueous solution is at least 0.01mg/ml.
 6. The method according to claim 4 wherein said surface isexposed to said aqueous solution containing said degradation enzyme fora period of at least one hour.
 7. The method according to claim 5wherein the concentration of said non-ionic surfactant in said aqueoussolution is at least 0.005 percent by weight.
 8. The method according toclaim 7 wherein said non-ionic surfactant is selected from the groupconsisting of Triton™, Span™, Tween™, and sucrose esters.
 9. The methodaccording to claim 7 wherein said non-ionic surfactant is Triton X-100.10. The method according to claim 1 wherein the degradation enzyme isselected from the group consisting of cutinases and lipases.
 11. Themethod according to claim 1 wherein said degradation enzyme is cutinaseisolated from Pseudomonas mendocina.
 12. A method for preparingdehydrated fruits and vegetables from unmacerated harvested fruits andvegetables wherein said harvested fruits and vegetables have a wateractivity (a_(w)) of greater than 0.80 and further have a surfacemembrane which contains one or more types of water insoluble componentsselected from the group consisting of cutin, cellulose, pectin,triglycerides and waxy esters, which method comprises:(a) exposing thesurface membrane of said harvested fruit or vegetable to a compositioncomprising a sufficient concentration of a degradation enzyme selectedfrom the group consisting of cutinase, cellulase, pectinase and lipaseand a non-ionic surfactant at a concentration sufficient to enhance theactivity of said degradation enzyme for a sufficient period so as toprovide for an increase in water permeability across said membrane of atleast fifty percent as compared to said fruits and vegetables prior toexposure to said composition; and (b) dehydrating the fruit or vegetableproduct produced by step (a) above under conditions sufficient to reduceits a_(w) to 0.80 or less.
 13. The method according to claim 12 whereinthe increase in water permeability across said membrane is at least onethousand percent.
 14. The method according to claim 12 wherein saiddegradation enzyme is contained in an aqueous solution.
 15. The methodaccording to claim 14 wherein the concentration of said degradationenzyme in said aqueous solution is at least 0.01 mg/ml.
 16. The methodaccording to claim 15 wherein said surface is exposed to said aqueoussolution containing said degradation enzyme for a period of at least onehour.
 17. The method according to claim 12 wherein the concentration ofsaid non-ionic surfactant in said aqueous solution is at least 0.005percent by weight.
 18. The method according to claim 12 wherein saidnon-ionic surfactant is selected from the group consisting of Triton™,Span™, Tween™, and sucrose esters.
 19. The method according to claim 12wherein said non-ionic surfactant is Triton X-100.
 20. The methodaccording to claim 12 wherein said degradation enzyme is selected fromthe group consisting of cutinases and lipases.
 21. The method accordingto claim 12 wherein said degradation enzyme is cutinase isolated fromPseudomonas mendocina.
 22. The method according to claim 12 wherein saiddehydrating step is selected from the group consisting of osmosis andevaporation.
 23. The method according to claim 22 wherein saiddehydrating step is an osmotic dehydration.
 24. The method according toclaim 23 which further comprises drying the dehydrated product producedin step (b) to remove at least one of the residual moisture.
 25. Amethod of transporting a natural or synthetic substance into theinterior of an unmacerated harvested fruit or vegetable having a surfacemembrane which contains one or more types of water insoluble componentsselected from the group consisting of cutin, cellulose, pectin,triglycerides and waxy esters, which method comprises:(a) exposing thesurface membrane of said fruit or vegetable to a composition comprisinga sufficient concentration of a degradation enzyme selected from thegroup consisting of cutinase, cellulase, pectinase, and lipase and asufficient concentration of a non-ionic surfactant so as to enhance theactivity of said degradation enzyme for a sufficient period of time soas to provide an increase in water permeability across said membrane ofat least 50 percent as compared to the water permeability of saidharvested fruit or vegetable prior to exposure to said composition; and(b) exposing the product produced by step (a) above with an aqueoussolution containing sufficient quantities of natural and/or syntheticsubstance(s) for a sufficient period of time so as to allow saidsubstance(s) to be osmotically transported to the interior of saidproduct.
 26. The method according to claim 25 wherein the increase inpermeability across said membrane is from at least 100 percent to about400 percent.
 27. The method according to claim 26 wherein saiddegradation enzyme is contained in an aqueous solution.
 28. The methodaccording to claim 27 wherein the concentration of said degradationenzyme in said aqueous solution is at least 0.01 mg/ml.
 29. The methodaccording to claim 28 wherein said surface is exposed to said aqueoussolution containing said degradation enzyme for a period of at least onehour.
 30. The method according to claim 28 wherein the concentration ofsaid non-ionic surfactant in said aqueous solution is at least 0.005percent by weight.
 31. The method according to claim 30 wherein saidnon-ionic surfactant is selected from the group consisting of Triton™,Span™, Tween™, and sucrose esters.
 32. The method according to claim 30wherein said non-ionic surfactant is Triton X-100.
 33. The methodaccording to claim 25 wherein said degradation enzyme is selected fromthe group consisting of cutinases and lipases.
 34. The method accordingto claim 25 wherein said degradation enzyme is cutinase isolated fromPseudomonas mendorina.
 35. The method according to claim 25 wherein saidsubstance is a natural substance.
 36. The method according to claim 25wherein said substance is a synthetic substance.